Expression of a Cryptic Secondary Sigma Factor Gene Unveils Natural Competence for DNA Transformation in Staphylococcus aureus
A) Overexpression of sigH mRNA is not sufficient for SigH activation. N315ex carrying pMKcomGFP and pRIT-sigHNH7 was grown in CS2, LB, or BHI medium aerobically at 37°C for indicated time periods, and analyzed by Western blot. Only growth in CS2 medium led to GFP production. B–C) An inverted repeat sequence in the sigH 5′-UTR negatively controls its expression. B) Sequences of the 5′-UTR region in each sigH-expressing plasmid. Arrows show the 13 bp inverted repeat, and the translation initiation codon is underlined. PRIT-sigHNH7 carries the native nucleotide sequence. The inverted repeat sequence was partially deleted in pRIT-sigHIRd, whereas it was entirely removed and the SD sequence replaced with a consensus ribosome binding site in pRIT-sigH. C) Deletion of one half of the inverted repeat allows all cells to produce active SigH. N315ex derivatives carrying the plasmids indicated on the left were grown in CS2 or TSB at 37°C with shaking. Note that all cells of N315ex carrying pRIT-sigHIRd show GFP signals, even in TSB. D) Northern blot analysis confirmed the accumulation of sigH mRNA in cells carrying all of the different sigH expression plasmids. N315ex cells carrying the designated plasmid together with pMKcomGFP, were grown in TSB medium. Exponentially growing cells (OD600 = 0.5) were used for Northern blot analysis as described in Materials and Methods. The lower panel shows the EtBr stained agarose gel as a loading control.